Activity levels of cathepsins B and L in tumor cells are a biomarker for efficacy of reovirus-mediated tumor cell killing.

Reovirus has gained much attention as an anticancer agent; however, the mechanism of the tumor cell-specific replication of reovirus is not fully understood. Although Ras activation is known to be crucial for tumor cell-specific replication of reovirus, it remains controversial which cellular factors are required for the reovirus-mediated tumor cell killing. In this study, we systematically investigated which cellular factors determined the efficiencies of reovirus-mediated tumor cell killing in various human cultured cell lines. The efficiency of reovirus-mediated cell killing varied widely among the cell lines. Junction adhesion molecule-A, a reovirus receptor, was highly expressed in almost all cell lines examined. Ras activation levels were largely different between the cell lines; however, there were no apparent correlations among the reovirus-mediated cell killing efficiencies and Ras activation status. On the other hand, activity levels of the cysteine proteases cathepsins B and L, which are crucial for proteolytic disassembly of the outer capsid proteins of reovirus, showed a tendency to be correlated with the efficiency of reovirus-mediated cell killing. These results indicate that the activity of cathepsins B and L is the most suitable as a biomarker for the efficacy of reovirus-mediated oncolysis among the factors examined in this study.

Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.

Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features.

Insect lysozyme from house fly (Musca domestica) larvae: possible digestive function based on sequence and enzymatic properties.

Lysozyme was purified from the homogenate of the whole body of house fly (Musca domestica) larvae by standard chromatographic techniques. The purified lysozyme was sequenced and its enzymatic properties were examined. This lysozyme was a chicken-type lysozyme composed of 122 amino acids, showing about 75% identity with fruit fly lysozymes and 38% with human lysozyme. This enzyme was inactive towards Micrococcus luteus and under the physiological conditions of PH 7.0 and ionic strength 0.1, but was as active toward glycol chitin as was hen lysozyme. The pH-dependent profile of lytic activity towards M. luteus showed that house fly lysozyme has an acidic pH optimum and shows no enzymatic activity above Ph 7. These features are analogous with those of ruminant stomach lysozymes which have evolved for the digestive function, suggesting that this lysozyme does not function as a self-defense protein, like hen and human lysozyme, but as a digestive enzyme, probably in the gut of the insect body. Although a similar functional conversion to digestive enzyme was reported in fruit fly, phylogenetic tree analysis indicates that the evolutionary change of lysozyme to a digestive enzyme occurred similarly in fruit fly and house fly, but the events are not related and occurred independently in each strain. This observation is in contrast with the case of ruminant stomach lysozymes, which were recruited before the divergence of each species of ruminants.

Antinociceptive effects of oriental medicine kei-kyoh-zoh-soh-oh-shin-bu-toh in mice and rats.

Antinociceptive effects of peroral administration of an oriental medicine Kei-Kyoh-Zoh-Soh-Oh-Shin-Bu-toh (TJ-8023) were examined using rats and mice. TJ-8023 (100 mg.kg-1.d-1) inhibited the induction of adjuvant-induced hyperalgesia of rats in the paw-pressure test following prophylactic administration, and normalized the nociceptive threshold at a dose of 600 mg.kg-1.d-1, without effects on adjuvant-induced inflammation. Mice suffering from repeated cold stress showed a decrease in nociceptive threshold for tail-pressure stimulation. Such a hyperalgesia was reversed by a single (30 and 100 mg/kg) or repeated administration (100 and 300 mg.kg-1.d-1) of TJ-8023. The nociceptive threshold of non-stressed mice was not affected by TJ-8023 (100 mg/kg). Nociceptive responses of mice to cold-plate stimulation were also not affected by repeated administration of TJ-8023 (300, 600 and 1200 mg.kg-1.d-1). The present results demonstrate the antinociceptive action of TJ-8023, and suggest that it is more effective in easing a hyperalgesia in morbid state than in suppressing a nociception in the normal one.